Modeling the Initiation Phase of the Catalytic Cycle in the Glycyl-Radical Enzyme Benzylsuccinate Synthase

The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.


EPR measurements
The EPR spectrum confirming H/D exchange in D 2 O at glycyl radical in BSS isolated from Aromatoleum sp. was recorded according to the following procedure.The X-band CW-EPR spectra were measured at 200 K using Bruker Elexsys E580 spectrometer and SHQ4122 resonator equipped with ESR900 cryostat (Oxford Instruments).Parameters were as follows: microwave frequency: 9.388 GHz; microwave power: 0.6 mW; modulation amplitude and frequency: 0.4 mT and 100 kHz, respectively; time constant: 20 ms; sweep time: 42 s; sweep width: 30 mT; center field: 334.6 mT.

Results
Fumarate binding pocket   The geometries of the stationary points Figure S1.Calibration curves for LC-MS/MS quantitation of A) LC-DAD quantitation of benzylsuccinate; B) benzylsuccinate in SIM mode.Concentrations are given in µg/L.
Figure S2.The QM (HL) part of the QM:MM BSS apoenzyme models used in the study of H 2 O-assisted radical transfer.S-HL for a) S-QM and b) B-HL.
Figure S3.The interpolated charge of the BSS protein depicted on the surface of the fumarate binding pocket.The blue regions indicate a positive charge while the red regions indicate a negative charge.The left blue region on the top view originates from Arg508 while the right blue region from the main chain of the Met404 and Cys494.The negative charge patch visible in the side view is located close to the protonated carboxyl group and comes from the carbonyl group of Asn615.
Figure S8.The geometry of stationary points obtained for E:S holoenzyme -re and si attack.
Figure S9.Geometry of stationary points obtained for E:P holoenzyme -re and si attack.
Figure S10.Geometry of stationary points obtained for apoenzyme -re and si attack.

Table S1
Parameters of jet-stream ESI ion source used in the LC-MS/MS analysis.

Table S2 .
Parameters of MRM method for the analysis of d 7 -benzylsuccinate and d 8 -benzylsuccinate.

Table S3 .
Parameters of Single Ion Monitoring (SIM) method for the analysis of D/H exchange in benzylsuccinate in D 2 O.

Table S6 .
Differences in electronic energies corrected by thermal energy calculated for the holoenzyme at different levels of theory.

Table S7 .
Electronic energies of the apoenzyme calculated for small and big high layer (S-QM and B-QM) and vibrational corrections calculated for

QM tzvp level of theory was
estimated based on an energy difference between TS apo and I at E B-QM dzvp level

Table S8 .
Differences in electronic energies corrected by thermal energy calculated for the apoenzyme at different levels of theory

E+thermal) S-QM tzvp [kJ/mol] (E+thermal) S-QM tzvp [kJ/mol]
Difference in electronic energy between TS apo and I at B-QM tzvp was estimated as the same as between TS apo and I at B-QM dzvp level of theory Prediction of elementary rate constants

Table S9 .
Kinetic rate constants and iKIE calculated for E:S complex model

Table S10 .
Kinetic rate constants and iKIE calculated for E:P complex model

Table S11 .
Kinetic rate constants and iKIE calculated for apoenzyme, E+Thermal)' takes into account the energy difference between proR and proS conformations,